Tuberculosis (TB) retains its place as a global scourge, killing an estimated 2 million people per year despite the availability of chemotherapy. The huge majority of cases are clinically latent and remain difficult to diagnose. Control of the global epidemic hinges on our ability to identify and treat this vast population of latently infected individuals;as such, it is critical that our fundamental view of chronic Mycobacterium tuberculosis (MTB) infection is accurate. However, despite a century of research, the physiologic state of MTB during persistent infection remains poorly understood. To monitor MTB replication within hosts, we have developed an unstable plasmid that is lost at a stable, quantifiable rate from dividing cells in the absence of antibiotic selection for use as a replication clock. With this tool, we have recently determined that MTB continues to replicate throughout chronic infection of mice and is restrained by the host immune system. This result argues for a thorough re-appraisal of current TB persistence models that postulate bacteriostasis. In addition, significant practical questions are raised, since TB drug discovery efforts increasingly focus on eliminating non-replicating organisms as the key to improving therapy and curing latent infection. It is therefore important to evaluate both the unstable plasmid replication clock and our assumptions about MTB latency as quickly and completely as possible. In this proposal, we outline a series of experiments to test our findings of MTB replication dynamics and expand our view of infection within a host. First, we will analyze key parameters of plasmid behavior both in vitro and in vivo to refine the application of our technique. This will involve use of environmental stressors as well as a conditionally replicating mutant strain to produce a robust model of plasmid segregation. Second, we will employ fluorescent protein technology in conjunction with the replication clock to visualize and quantify MTB dynamics at the level of the granuloma. This innovation will allow us to characterize the heterogeneity of bacterial replication in different lesions within a single infected organ. Completion of our proposed studies will firmly establish a unique tool with which to evaluate MTB behavior within a host. This technique promises not only significant insight into key aspects of MTB latency, but offers application within larger efforts of therapeutic discovery and vaccine development. PUBLIC HEALTH RELEVANCE: Current knowledge of the host and bacterial factors that are involved in latent tuberculosis (TB) are woefully inadequate. This project will generate novel tools and insights to improve our understanding of M. tuberculosis dynamics in vivo. The result will be a vastly more accurate picture of TB latency and pathogenesis, which will in turn spark efforts to combat this scourge that afflicts two billion people in the world today.